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Abstract

Trypanosomes undergo deep morphological changes when they transform from proliferative forms into infective forms. These changes are especially noticeable in the nucleus and correlate with decrease in the transcriptional rate. The nucleus of proliferative forms is round, contains a large nucleolus and small amounts of peripheral heterochromatin. The nucleus of trypomastigotes forms is elongated, the nucleolus disappears, and the heterochromatin occupies most of the nuclear space. As chromatin packing is related to transcription, we compared the chromatin structure in epimastigote and trypomastigote forms. Nuclei obtained from parasites disrupted by nitrogen cavitation remains intact and resemble the structure found in intact parasites. Treatment of the isolated nuclei with Microccocal nuclease reveals a typical nucleosome structure as previously described. However, nuclei obtained from trypomastigotes were more susceptible to digestion with Microccocal nuclease than epimastigote nuclei, although a larger part of trypomastigote DNA is totally resistant. This finding suggest that in trypomastigotes, most of the genome is arranged in a freely and accessible nucleosome structure, and part of it is complexed in a highly resistant domain, probably related to large amounts of heterochromatic material. In epimastigotes, the nucleosomal structure is probably protected from cleavage by the presence of the transcription and RNA processing machinery. In addition, we found that the total histones isolated from the purified nuclei differ in these life stages, which might be due to differential histone modifications or expression. Northern blots show that at least histone H1 is underexpressed in epimastigotes. Therefore, the chromatin may be differentially organized in proliferative and infective stages of Trypanosoma cruzi. Two hot start PCR procedures for amplification of Trypanosoma cruzi kinetoplastid 330 bp DNA and nuclear satelite 188 bp DNA were optimized for detection of T. cruzi in blood samples. Both PCR procedures were prospectively evaluated in a pediatric population followed by the Chagas service of the Ricardo Gutierrez Children's Hospital in Buenos Aires. Hereby we analize the first 32 evaluated clinical cases, including 25 children from chagasic mothers, 3 children with toxoplasmosis, 4 normal children as controls. Clinical diagnosis of Chagas disease was assessed by microhematocrite in children younger than six months old (Group A, n = 10) and by conventional serology in older children and mothers (Groups B and C respectively). 2 ml of peripheral blood were withdrawn from group A; and 10 ml of blood from the other clinical groups. Blood was diluted in one volume of Guanidinechloride-EDTA solution and stored at 4°C. Sera from all patients were also taken for conventional diagnosis and ELISA with recombinant antigens. PCR and recombinant ELISA were done blindly, results from the first 38 cases were finally compared with clinical diagnosis. Recombinant ELISA and conventional serology had 100% of correlation . Out...